Attune nxt flow cytometer software

For analysis of mambalgin-2’s influence on the cell cycle, the A549 and metastatic Lewis cells were seeded in six-well culture plates (2 × 10⁵ cells per well) at pH 5.5, grown for 24 h, and incubated with 1 μM mambalgin-2 for 72 h without the media change. Then the cells were detached from the wells by 0.5% trypsin-EDTA, washed with Earle’s Balanced Salt Solution (EBSS), and fixed in ice-cold 70% ethanol for 24 h at -20°C. After fixation, the cells were washed twice with EBSS, and DNA was extracted by 5 min of incubation with the DNA extraction buffer (200 mM Na₂HPO₄ with 0.004% Triton X-100, pH 7.8). Then the cells were washed with EBSS, resuspended in the DNA staining solution (EBSS, 50 mg/ml propidium iodide, 0.2 mg/mL DNAse-free RNAse A), and analyzed using the Attune NxT flow cytometer (Life Technologies). The data were analyzed using the Attune NxT flow cytometer Software (Life Technologies).

For analysis of the acidification influence on the cell cycle, the mel P cells were seeded in 6-well culture plates (2 × 10⁵ cells per well) in the normal or acidic medium and grown for 96 h with the media change every 48 h. For analysis of the mambalgin-2 influence on the cell cycle, mel P cells were seeded in 6-well culture plates (2 × 10⁵ cells per well) in the acidic medium, grown for 24 h, and incubated with 1 μM mambalgin-2 for 72 h without the media change. Then the cells were detached from the wells by 0.5% trypsin-EDTA, washed with Earl balanced salt solution (EBSS), and fixed in ice-cold 70% ethanol for 24 h at −20 °C. After fixation, the cells were washed twice by EBSS, and DNA was extracted by 5 min incubation with the DNA extraction buffer (200 mM Na₂HPO₄ with 0.004% Triton X-100, pH 7.8). Then the cells were washed with EBSS, resuspended in the DNA staining solution (EBSS, 50 mg/mL propidium iodide, 0.2 mg/mL DNAse free RNAse A), and analyzed by the Attune NxT flow cytometer (Life Technologies, Carlsbad, CA, USA). The data were analyzed using the Attune NxT flow cytometer Software (Life Technologies).

Intracellular cytokine staining of T cells was performed after stimulation with 50 ng/ml phorbol myrisate acetate (PMA) (Enzo Life Sciences AG), 1 μg/ml Ionomycin (Enzo Life Sciences AG), and 6.6 μl/10 ml Golgi stop (BD Biosciences) for 4 h before harvesting the T cells. T cells were then fixed with Cytofix buffer (BD Biosciences) for 15 min at 20°C, washed with PBS, permeabilized with perm/wash buffer (BD Biosciences) for 15 min at 20°C and stained with fluorophore-conjugated mAbs and the respective isotype control mAbs. Flow cytometry was performed on a FACSCalibur using CellQuest software (BD Biosciences) or Attune NxT using Attune NxT Flow Cytometer software (Thermo Fisher Scientific) and analysis was done with FlowJo software (Version 10).